Chlamydia trachomatis is a leading cause of sexually transmitted disease (STD) and trachoma the worlds leading cause of blindness. C. trachomatis isolates are separated into biovariants depending on the disease they cause. The trachoma biovars produce localized infection of the eye or genital mucosa. Infections with these strains are a major cause of STD and are characterized by persistent infection of mucosal surfaces resulting in chronic inflammatory disease. In women these infections are manifested as pelvic inflammatory disease whose sequelae can produce tubal blockage and infertility as well as tubal pregnancy. In contrast, the non-trachoma strains cause lymphogranuloma venereum (LGV) an invasive STD. LGV strains transiently infect the genital mucosa but then rapidly disseminate to the draining lymph nodes where the parasite infects monocytes and granuloma formation. An understanding of the molecular basis for these marked differences in virulence and pathogenesis are likely key to the identification of new parasite molecules that can be targeted for novel intervention strategies against chlamydial disease. The genomes of a trachoma-STD and a LGV biovarinat have recently been sequenced and annotated. Comparative genomic analysis showed remarkable similarity of the small1-1.2 million base pair genomes of each biovarinat despite their marked differences in tissue tropism and disease manifestation. However, a single gene family (CT 166-169) with homology to the large clostridial cytotoxin toxin B was found in the trachoma-STD biovariants genome which was not present in the LGV genome. It is therefore possible that this putative chlamydial toxin plays a critical role in the virulence and pathogenesis of the trachoma-STD biovariants. We have therefore focused this project on studies to define whether the trachoma-STD variants express a biologically active toxin and if so what role the toxin plays in the pathogenesis of disease caused by these medically important pathogens. Our first goal was to document that trachoma-STD biovariants and not LGV strains express an active toxin. We found that inoculation of HeLa 229 cells with a high multiplicity of infection of a trachoma-STD strain produced an immediate cytotoxicity characterized by cell rounding and actin depolymerization. RT-PCR analysis of cells at different time periods post-chlamydial infection detected CT166 mRNA in mid to late cycle infected cells indicating the CT166 is a late gene product. Hyperimmune rabbit antiserum was generated against recombinant CT166 and synthetic peptides corresponding to primary CT166 sequence. These antisera were used to probe western blots of purified chlamydial organisms and infected cells. We found an immunoreactive 350 kDa polypeptide in lysates of purified elementary bodies and infected HeLa 229 cells of a trachoma-STD strain but not in similar antigen preparations made from a LGV strain. These findings are the first to demonstrate a biologically active toxin for C. trachomatis strains associated with chronic diseases of the oculogenital mucosa.